The present invention relates to the field of glycosyltransferases, or enzymes which transfer sugar residues from an activated donor substrate to an amino acid or growing carbohydrate group.
Glycosyltransferases that are involved in the biosynthesis of glycoprotein and glycolipid sugar chains are resident membrane proteins of the endoplasmic reticulum and the Golgi apparatus. They are responsible for catalysis of the addition of monosaccharide units either to an existing glycan chain or to a peptide or lipid acceptor initiating a chain. Donor monosaccharides are typically utilized in activated form, either as a nucleotide sugar, e.g. GDP-mannose or, less frequently, as a lipid-linked donor, e.g., dolichol-P-glucose (Dol-P-Glc). The majority of glycosyltransferases are lumenally oriented, i.e. with the catalytic domain within a membrane-bounded compartment. Examples of lumenally oriented enzymes are galactosyltransferases and sialyltransferases. Their structure is pictorially represented in FIG. 1. The enzymes are typically grouped into families based on the type of sugar they transfer (galactosyltransferases, sialyltransferases, etc.). Comparisons amongst known cDNA clones of glycosyltransferases (Paulson, J. C. & Colley, K. J., J. Biol. Chem. 264 (30), 17615-618 (1989), has revealed that there is very little sequence homology between the enzymes. However, as indicated by FIG. 1, all glycosyltransferases share some common structural features: a short NH.sub.2 -terminal cytoplasmic tail, a 16-20 amino acid signal-anchor domain, and an extended stem region which is followed by the large COOH-terminal catalytic domain. The signal anchor domains act as both uncleavable signal peptides and as membrane-spanning regions and orient the catalytic domains of these glycosyltrarisferases within the lumen of the Golgi apparatus.
The means by which cells regulate the expression of specific carbohydrate sequences is of great interest because of increasing evidence that cell surface carbohydrate groups mediate a variety of cellular interactions during development, differentiation, and oncogenic transformation. von Figura, K. & Hasilik, A., Annu. Rev. Biochem. 55, 167-193 (1986); Komfield, S., J. Clin. Invest. 77, 1-6 (1986); Munro, S. & Pelham, H.R.B., Cell 48, 899-907 (1987); Pelham, H. R. B., EMBO J. 7, 913-918 (1988); Paabo, S. et al., Cell 50, 311-317 (1987). It is estimated that at least one hundred (100) glycosyltransferases are required for the synthesis of known carbohydrate structures on glycoproteins and glycolipids, and most of these are involved in elaborating the highly diverse terminal sequences. Paulson, J. C. & Colley, K. J., J. Biol. Chem. 264 (30), 17615-618 (1989). Among those enzymes responsible for terminal elaborations, three (3) enzymes have been of particular interest: galactosyltransfereases, fucosyltransferases and sialyltransferases.
Fucosyltransferases transfer the sugar fucose from UDP in .alpha.1-2, .alpha.1-3, .alpha.1-4 and .alpha.1-6 linkages. Fucose was first identified as being present in glycosidic linkages to serine or threonine as compounds of the type Glcb1.fwdarw.3Fuca1.fwdarw.O-Ser/Thr and Fuca1.fwdarw.O-Ser/Thr in human urine and rat tissue. Hallgren, P. et al., J. Biol. Chem. 250, 5312-5314 (1975); Klinger, M. M. et al., J. Biol. Chem. 256, 7932-7935 (1981). The identification of O-linked fucose attached to a specific protein was first made by Kentzer at al. who found a residue of fucose covalently linked to a peptide derived from the epidermal growth factor (EGF) domain of recombinant urokinase. Kentzer, E. J. et al., Biochem. Biophys. Res. Commun., 171, 401-406 (1990). Similar glycosylation patterns have been found in tissue plasminogen activator (tPA) (Harris, R. J. & Spellman, M. W., Biochemistry 30, 2311-14 (1991)), human factor VII (Bjoern et al. 266, 11051-11057 (1991)), human factor XII, (Harris et al., J. Biol. Chem., 267, 5102-5107 (1992)) and vampire bat plasminogen activator, Gardell et al, J. Biol. Chem. 264, 17947-52 (1989). The EGF domain of human factor IX has also been indicated to have O-fucosylation, but at the reducing end of the tetrasaccharide: NeuAca2.fwdarw.6Galb1.fwdarw.4GlcNAcb1.fwdarw.3Fuca1.fwdarw.O-Ser61. Nishimura et al., J. Biol. Chem., 267, 17520-17525 (1992); Harris et al., Glycobiology 3, 219-224 (1993). However, in all cases in which it has been detected, O-linked fucose is present within the sequence Cys-Xaa-Xaa-Gly-Gly-Ser/Thr-Cys. Harris et al., Glycobiology 3, 219-224 (1993).
EGF is a potent 53 amino acid mitogen which has its activity mediated by binding to the EGF receptor. Carpenter, G and Cohen, C, J. Biol. Chem. 265, 7709-7712 (1990). Regions of EGF sequence homology have been found in an every-increasing number of coagulation, fibrinolytic, complement and receptor proteins. Panthy, L., FEBS Lett. 214, 1-7 (1987); Doolittle, R. F., Trends Biochem. Sci. 14, 244-245 (1989). The EGF modules of these multi-modular proteins are not believed to interact with the EGF receptor. Rather, different properties have been ascribed to such EGF modules, including ligand binding (Appella et al., J. Biol. Chem. 262, 4437-4440 (1987); Kurosawa et al., J. Biol. Chem. 263, 5993-5996 (1988), mitogenic activity (Engel, FEBS Lett. 251, 1-7 (1989) and receptor recycling (Davis et al., Nature 326, 760-765 (1987). The EGF modules of the vitamin K-dependent coagulation proteins are required for the proper folding of adjacent modules containing .gamma.-carboxylglutamic acid residues (Astermark et al., J. Biol. Chem. 266, 2430-2437 (1991), while others may simply serve as spacers between different functionally active regions (Stenflo, J., Blood 78, 1637-1651 (1991).
EGF domains are characterized by the presence of six (6) conserved cysteine residues that are expected to form three (3) intrachain disulfide bonds in the 1-3, 2-4 and 5-6 pattern obtained for EGF. Savage et al., J. Biol. Chem. 248, 7669-7672 (1973). A similar disulfide-binding pattern has been confirmed for the EGF domain of human complement protein Cls, Hess et al., Biochemistry 30, 2827-2833 (1991). Three dimensional solution structures of synthetic comprising individual N-terminal EGF modules of human factors X and IX have been obtained by NMR spectroscopic studies (Selander et al., Biochemistry 29, 8111-8118 (1990); Huang et al., Biochemistry 30, 7402-7409 (1991); Baron et al., Protein Sci. 1, 81-90 (1992); Ullner et al., Biochemistry 31, 5974-5983 (1992). The derived structures are almost identical to those determined for EGF (Cooke et al, Nature 327, 339-341 (1987) and TGF-.alpha. (Kohda et al, Biochemistry 28, 953-958 (1989); Tappin et al., Eur. J. Biochem. 179, 629-637 (1989).
There is an intense interest in the synthesis of proteins which contain O-fucose in glycosidic linkages. This is especially true in proteins with EGF domains which are O-fucosylated. In order to properly and efficiently O-fucosylate these proteins, an enzyme specific to creating O-fucose linkages would be highly desirable. However, as previous attempts to isolate and purify O-fucosyltransferase have proved to be unsuccessful, there exists a great need for highly pure, homogeneous O-fucosyltransferase as well as an efficient detection assay.